Intracellular Ca2+ increase induces post-fertilization events via MAP kinase dephosphorylation in eggs of the hydrozoan jellyfish Cladonema pacificum

Developmental Biology 293 (2006) 228 – 241 www.elsevier.com/locate/ydbio

Intracellular Ca 2+ increase induces post-fertilization events via MAP kinase dephosphorylation in eggs of the hydrozoan jellyfish Cladonema pacificum Eri Kondoh a , Kazunori Tachibana b , Ryusaku Deguchi a,⁎ a

b

Department of Biology, Miyagi University of Education, Aoba-ku, Sendai, Miyagi 980-0845, Japan Laboratory of Cell and Developmental Biology, Graduate School of Bioscience, Tokyo Institute of Technology, Midori-ku, Yokohama, Kanagawa 226-8501, Japan Received for publication 4 October 2005; revised 3 February 2006; accepted 3 February 2006 Available online 10 March 2006

Abstract Naturally spawned eggs of the hydrozoan jellyfish Cladonema pacificum are arrested at G1-like pronuclear stage until fertilization. Fertilized eggs of Cladonema undergo a series of post-fertilization events, including loss of sperm-attracting ability, expression of adhesive materials on the egg surface, and initiation of cell cycle leading to DNA synthesis and cleavage. Here, we investigate whether these events are regulated by changes in intracellular Ca2+ concentration and mitogen-activated protein kinase (MAP kinase) activity in Cladonema eggs. We found that MAP kinase is maintained in the phosphorylated form in unfertilized eggs. Initiation of sperm-induced Ca2+ increase, which is the first sign of fertilization, was immediately followed by MAP kinase dephosphorylation within a few minutes of fertilization. The fertilized eggs typically stopped sperm attraction by an additional 5 min and became sticky around this time. They further underwent cytokinesis yielding 2-cell embryos at ∼1 h post-fertilization, which was preceded by DNA synthesis evidenced by BrdU incorporation into the nuclei. Injection of inositol 1,4,5trisphosphate (IP3) into unfertilized eggs, which produced a Ca2+ increase similar to that seen at fertilization, triggered MAP kinase dephosphorylation and the above post-fertilization events without insemination. Conversely, injection of BAPTA/Ca2+ into fertilized eggs at ∼10 s after the initiation of Ca2+ increase immediately lowered the elevating Ca2+ level and inhibited the subsequent post-fertilization events. Treatment with U0126, an inhibitor of MAP kinase kinase (MEK), triggered the post-fertilization events in unfertilized eggs, where MAP kinase dephosphorylation but not Ca2+ increase was generated. Conversely, preinjection of the glutathione S-transferase (GST) fusion protein of MAP kinase kinase kinase (Mos), which maintained the phosphorylated state of MAP kinase, blocked the post-fertilization events in fertilized eggs without preventing a Ca2+ increase. These results strongly suggest that all of the three post-fertilization events, cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle, lie downstream of MAP kinase dephosphorylation that is triggered by a Ca2+ increase. © 2006 Elsevier Inc. All rights reserved. Keywords: Intracellular calcium increase; Mitogen-activated protein kinase; Sperm attraction; Surface adhesion; Cell cycle; IP3; BAPTA; U0126; Mos; BrdU

Introduction Marine hydrozoan jellyfish, as well as freshwater hydra, belong to Hydrozoa, one class of the evolutionarily old diploblastic phylum Cnidaria (Philippe et al., 1994; Collins, 2002; Gilbert, 2003). As is the case for higher triploblastic animals, hydrozoan eggs display an intracellular Ca2+ increase at fertilization (reviewed by Stricker, 1999). The Ca2+ increase is initiated by a Ca2+ wave, which starts from the animal pole (the only site of sperm-egg fusion in hydrozoan eggs), and ⁎ Corresponding author. Fax: +81 22 211 5791. E-mail address: [email protected] (R. Deguchi). 0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.02.005

propagates in the eggs toward the antipode (Deguchi et al., 2005). It seems likely that the Ca2+ increase is mainly due to a Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores (Freeman and Ridgway, 1991; Deguchi et al., 2005). These data support the view that the Ca2+-mobilizing system in hydrozoan eggs is very similar to that in more advanced animals such as sea urchins, starfish, frogs, and mammals (see Deguchi et al., 2005). In hydrozoans, sperm chemotaxis toward eggs is thought to play an important role in elevating the rate of fertilization. The sperm-attracting substances in hydrozoans are assumed as species-specific peptides with molecular weights of 2–25 kDa (Miller, 1985; Cosson et al., 1986). Except in some groups such

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as siphonophores (Carré and Sardet, 1981; Cosson et al., 1986), the sources of the substances may be the eggs themselves in most hydrozoan species (Miller, 1978, 1979; Freeman, 1987; Freeman and Miller, 1982). In such hydrozoans, the spermattracting ability, which is prominent in unfertilized eggs, declines and eventually vanishes after fertilization (Miller, 1978; Freeman and Miller, 1982). The loss of the spermattracting ability is artificially induced in unfertilized eggs by application of the Ca2+ ionophore A23187 (Freeman and Miller, 1982), which can trigger an intracellular Ca2+ increase similar to that seen at fertilization (Freeman and Ridgway, 1993). However, precise relationship between the Ca2+ increase and the cessation of sperm attraction has not yet been investigated in hydrozoan eggs. In some hydrozoans including Cladonema, eggs become sticky following fertilization (Hirai and Kakinuma, 1957; Yamashita, 1987). It seems likely that due to this phenomenon, fertilized Cladonema eggs can stay in the appropriate habitat, where Sargassum and other algae are abundant (Hirai and Kakinuma, 1957). The increase in the adhesive property in fertilized eggs suggests the secretion or transport of adhesive materials onto the egg surface, although Cladonema eggs contain no obvious cortical granules and thus exhibit no cortical reaction after fertilization (Yamashita, 1987). It is completely unknown whether the expression of adhesive property lies downstream of Ca2+ increase at fertilization. Unfertilized eggs of hydrozoans are arrested at G1-like pronuclear stage (Masui, 1985). Fertilization triggers release from the G1-arrest and onset of DNA synthesis in hydrozoan eggs. DNA synthesis is followed by nuclear envelope breakdown, separation of chromosomes, and cytokinesis yielding 2-cell embryos (Freeman and Ridgway, 1993). Similar cell cycle progression can be triggered by an A23187-induced Ca2+ increase; the artificially activated eggs undergo DNA synthesis and nuclear envelope breakdown, but resulted in irregular cytokinesis termed “pseudocleavage”, instead of normal cleavage (Freeman and Ridgway, 1993). This is the only available information about the involvement of Ca2+ increase in the cell cycle regulation in hydrozoan eggs. Extracellular signal-regulated kinases (ERKs), the members of mitogen-activated protein kinases (MAP kinases), are highly conserved from yeast to mammals (Kültz, 1998; English et al., 1999; Widmann et al., 1999). It has been shown that ERKs act at several steps in the cell cycle regulation (for recent reviews, see Roovers and Assoian, 2000; Abrieu et al., 2001; Zhang and Liu, 2002; Tunquist and Maller, 2003; Fan and Sun, 2004; Kishimoto, 2004), and their activities are sometimes regulated by intracellular Ca2+ levels (reviewed by English et al., 1999; Agell et al., 2002; Tunquist and Maller, 2003). In sea urchin eggs, which are arrested at G1-like stage until fertilization as is the case in hydrozoan eggs, G1/S transition is thought to occur through the control of ERK-like MAP kinase activity that is triggered by an intracellular Ca2+ increase (Carroll et al., 2000; Kumano et al., 2001; Philipova et al., 2005). In contrast, cortical granule exocytosis responsible for the formation of fertilization membrane, which is another main event caused by a Ca2+ increase at fertilization, seems to take place independently of the

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MAP kinase activity (Carroll et al., 2000). These data from sea urchin eggs prompted us to examine whether Ca2+ and/or MAP kinase are responsible for the post-fertilization events, cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle, in hydrozoan eggs. Here, we present evidence to indicate that MAP kinase is immediately dephosphorylated following fertilization as a result of Ca2+ increase in eggs of Cladonema pacificum. Our results also suggest that the MAP kinase dephosphorylation is necessary and sufficient for the induction of the post-fertilization events in Cladonema eggs. Materials and methods Animals and gametes The entire life cycle of C. pacificum can be regulated in our laboratory, which enables us to obtain mature jellyfish of this species throughout the year (Deguchi et al., 2005). Spawning was induced by placing the jellyfish under the dark for 20–25 min; mature eggs and sperm were released shortly after the dark period under our experimental condition of 20–22°C. Only freshly obtained eggs within 30 min of spawning were used because of their short-lived ability for normal fertilization (Deguchi et al., 2005). In contrast, the fertilizing ability of sperm could be preserved for up to 4 h when sperm suspension was maintained in a plastic culture dish coated with 10 mg/ml bovine serum albumin (BSA, fraction V, Sigma) at 20–22°C.

Ca2+ analysis The methods for Ca2+ recording and data analysis have been described in detail elsewhere (Deguchi and Morisawa, 2003; Deguchi et al., 2000, 2005). In brief, unfertilized eggs in filtered seawater (FSW) were injected with 100 μM Calcium Green-1 10-kDa dextran (CGD, Molecular Probes) dissolved in PAH buffer (100 mM potassium aspartate and 10 mM HEPES, pH 7.0). One or several dye-injected eggs were introduced into a measurement chamber, where they were slightly compressed by two coverslips coated with 10 mg/ml BSA. Fluorescence images of the targeted eggs in the chamber were captured with a silicon-intensified target tube (SIT) camera (C-2400, Hamamatsu Photonics) and continuously recorded on videotape. The fluorescence images on videotape were converted into digital images and processed using NIH Image (a public domain image processing software for the Macintosh computer). Insemination to unfertilized eggs was achieved by adding sperm suspension to a chamber, giving a final sperm concentration of 105–106/ml. To produce a Ca2+ increase similar to that seen at fertilization, 10 μM IP3 (Dojindo) prepared in PAH buffer was injected into unfertilized eggs. Conversely, a sperm-induced Ca2+ increase was suppressed by injecting BAPTA/Ca2+ (400 mM BAPTA and 80 mM CaCl2 in PAH buffer) into fertilized eggs. In these cases, the tip of pipette was inserted into deeper cytoplasm, and the chemicals were expelled from it; the estimated final concentrations of the injected chemicals in the cytoplasm ranged from 4 to 8% of the original concentrations in pipettes.

MAP kinase analysis U0126 (Promega), an inhibitor of MAP kinase kinase (MEK), and its inactive analogue, U0124 (Calbiochem), were prepared in DMSO at 5 mM and stored at −30°C. These reagents were diluted in FSW (final concentration: 20 or 100 μM) just before application to unfertilized eggs. The glutathione Stransferase (GST) fusion protein of starfish MAP kinase kinase kinase (Mos) and the control protein (GST alone) were prepared as described previously (Tachibana et al., 2000) and injected into unfertilized eggs together with CGD. The intracellular concentrations of these proteins were estimated to be 20– 40 μg/ml. The GST-Mos- and GST-injected eggs were inseminated at 3–5 min after injection. For immunoblotting, samples (each of which contained a lysate of 20 or 200 eggs depending upon experiments) were separated on 12.5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (BioTrace NT, Pall).

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Membranes were blocked for 30 min with TBS supplemented with 0.05% Tween-20 (TBST) and 5% skim milk (Difco) and subsequently incubated with each primary antibody, rabbit polyclonal anti-ERK1/2, CT antibody (Upstate Biotechnology) or rabbit polyclonal anti-phosphorylated ERK1/2 antibody (Sigma) at a dilution of 1:1000 for overnight at 4°C. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham) at a dilution of 1:10,000 for 1 h at room temperature. After washing with TBST, the membrane was incubated with ECL Advance Western blotting detection reagents (Amersham) for 5 min at room temperature, followed by exposure to luminescent image analysis (LAS1000 plus, Fuji) to obtain the luminescent image. Protein bands were analyzed by densitometry using Image Gauge Ver 3.4 (Fuji Film).

Sperm attraction To monitor the change in the egg's ability for sperm attraction, sperm behavior around a targeted egg in a chamber was viewed by a phasecontrast microscope and recorded on videotape via the SIT camera described above. In the image displayed on a screen (approximately 1000× magnification), a 5 × 5 cm square zone was set 2–3 cm away from the targeted egg, and the number of motile sperm crossing the sides of the square to enter the inner zone was continually scored per 10 s by slowmotion replay of video tape. During the long-term data recording (e.g., Fig. 2A), sperm suspension was repeatedly (every ∼2 min) added to the chamber, and the overflow was discarded accordingly, in order to compensate the loss of motile sperm due to their adhesion to the egg surface or coverslips. When the sperm-attracting ability was assessed for the egg that had been subjected to some experimental operation (e.g., IP3 injection), the targeted egg was transferred into a new chamber, and the number of “attracted” sperm was similarly scored every 10 s following insemination. In this case, the maximum number of attracted sperm during a recording period of 3 min was determined and used for the comparison of the sperm-attracting ability between the experimental and the control eggs (e.g., Fig. 3C).

Surface adhesion To examine whether or not surface adhesive property was expressed, a targeted egg was touched with a tungsten wire (100 μm in diameter) for 2–3 s under a stereomicroscope. Those eggs that stuck to the wire were regarded as adhesion positive.

Progression of cell cycle In hydrozoans, the activated eggs that have undergone DNA synthesis undergo cytokinesis independently of sperm penetration; normally fertilized eggs and artificially activated eggs without insemination exhibit normal cleavage and pseudocleavage, respectively (see Introduction). Thus, the occurrence of cytokinesis was used as morphological criteria for the progression of cell cycle. In some experiments, indirect immunofluorescence using anti-BrdU antibody was also performed to detect DNA synthesis. Eggs inseminated or stimulated with some reagent (e.g., IP3) were bathed in FSW containing 10 mM BrdU (Sigma). Following the culture in the continuous presence of BrdU, these eggs were fixed with 10% formaldehyde in FSW for 1 h. They were then washed three times with PBS by successively transferring them into fresh medium. The three washes with PBS were regularly performed between the two of the following operations. The fixed eggs were permeabilized with 1% Triton X-100 (Sigma) in deionized water for 1 h and then incubated in 4 M HCl for 1 h to denature the DNA. Incorporated BrdU in the nuclei was detected by sequential incubation with mouse anti-BrdU monoclonal antibody (Sigma) diluted 1:10 in PBS for 1 h and FITC-conjugated goat anti-mouse IgG (Sigma) diluted 1:100 in PBS for 1 h at room temperature. The specimens were finally mounted in a drop of glycerol containing 10% Tris–HCl (10 mM, pH 8.0) and 1% 1,4-diazabicyclo [2.2.2]octane (DABCO, Sigma), an anti-fluorescence bleaching agent. Only the specimens without showing non-specific labeling of the whole egg surface were selected for the observation.

Results Intracellular Ca2+ increase and MAP kinase dephosphorylation in normally fertilized eggs Insemination to CGD-injected Cladonema eggs caused an intracellular Ca2+ increase lasting for several minutes (n = 8; Fig. 1A). In all cases, the rising phase of the Ca2+ increase took the form of a Ca2+ wave starting from one cortical region and propagating to its antipode (data not shown). These spatiotemporal characteristics of sperm-induced Ca2+ increase were essentially the same as those reported previously (Deguchi et al., 2005). The time required from insemination to the first detectable Ca2+ increase ranged from 30 to 120 s (51 ± 27 s, mean ± SD). Thus, we estimated the time required from insemination to fertilization to be 1 min, in cases where the time of fertilization was not directly confirmed by Ca2+ signals. In unfertilized Cladonema eggs, polyclonal antibodies against pan-ERK and phosphorylated ERK (active form) both recognized only a single band of 42 kDa (Fig. 1Ba), which corresponds to the molecular mass of mammalian ERK2 (Kültz, 1998). Since a similar single ERK-like MAP kinase with the molecular mass of 42–44 kDa has been reported in oocytes or eggs of other marine invertebrates (e.g., Shibuya et al., 1992; Carroll et al., 2000; Stephano and Gould, 2000; Smythe and Stricker, 2004; Tan et al., 2005), we regarded the 42-kDa protein as an ERK-like MAP kinase in Cladonema eggs. The density of the band detected by the antibody against pan-ERK remained relatively constant during a period of 5 min following insemination (n = 4; Fig. 1Bb). In contrast, the staining for the antibody against phosphorylated ERK abruptly decreased at 2– 3 min post-insemination and almost completely disappeared within 5 min of insemination (n = 4; Fig. 1Bb). These results suggest that the 42-kDa MAP kinase in Cladonema eggs is maintained in the active form before fertilization but immediately inactivated after fertilization. The phosphorylated form of MAP kinase could not be detected in the later samples at least by the time of first cleavage (data not shown). Cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle in normally fertilized eggs To monitor the change in the egg's ability for sperm attraction after fertilization, we adopted a simplified method using phase-contrast microscopy and video recording; the number of motile sperm entering a fixed area near a targeted egg was calculated every 10 s (see Materials and methods), and this value was considered as the number of “attracted” sperm. Following insemination, the number of attracted sperm rapidly increased and reached the greatest value of ∼250 in the case of the egg for Fig. 2A. The considerable sperm-attracting ability was maintained for a few minutes; however, the number of attracted sperm gradually decreased subsequently and reached a sustained low level within 7 min of insemination (Fig. 2A) in spite of continuous addition of sperm suspension. Similar attenuation of sperm attraction was observed in the other 3 separate experiments with eggs from different females, although

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Fig. 1. Temporal patterns of intracellular Ca2+ increase (A) and MAP kinase dephosphorylation (B) at fertilization. (A) The CGD-injected egg was inseminated at time zero for monitoring the Ca2+ change at fertilization. The egg exhibited normal cleavage after the Ca2+ recording. (B) Lysates of eggs before and after insemination were separated on a 12.5% SDS-PAGE gel, transferred onto nitrocellulose membranes, and probed with polyclonal antibodies against pan-ERK (lane 1 in Ba and upper panel in Bb) and phosphorylated ERK (lane 2 in Ba and lower panel in Bb). Molecular weight makers are indicated in kilodalton. We regarded the 42-kDa protein recognized by the two antibodies (lanes 1 and 2 in Ba) as an ERK-like MAP kinase in Cladonema eggs. The protein bands in Bb were analyzed by densitometry. Relative levels of MAP kinase (open circles) and phosphorylated MAP kinase (filled squares) normalized by the values for unfertilized eggs (at time zero) are indicated.

the maximum number of attracted sperm was somewhat different in respective experiments (data not shown). A decrease in the sperm-attracting ability after fertilization was also confirmed in the eggs where the time of fertilization had been confirmed by monitoring the Ca2+ change. When the maximum number of attracted sperm was examined at 10 min post-fertilization, the value for the fertilized egg was considerably and always lower than that for the control egg, which had been injected with CGD similarly but kept unfertilized for 10 min before sperm attraction assay (n = 4; data not shown). These results indicate that the sperm-attracting ability has been lost within several minutes of fertilization in Cladonema eggs. As mentioned in the Introduction, it is known that Cladonema eggs become sticky after fertilization (Yamashita, 1987). To know the time course of the expression of surface adhesive property in fertilized eggs, we checked whether the eggs in which the time of fertilization has been confirmed by a

Ca2+ increase can adhere to a tungsten wire. At 3 min of fertilization, no adhesion to the tungsten wire was observed in any egg (Fig. 2B). However, the number of eggs showing adhesive property increased subsequently and reached the maximum value by 10 min post-fertilization (Fig. 2B). The data from the above two experiments (Figs. 2A and B) imply that the adhesive property appears around the time when the spermattracting ability has vanished. For the analysis of cell cycle, one advantage of using Cladonema eggs was high reliability of synchronous fertilization (see above). In addition, the rate of normal development was also high in this species; most of the fertilized eggs underwent cytokinesis and reached normal 2-cell embryos at ∼1 h post-fertilization (Fig. 2C). Immunofluorescence experiments for the detection of BrdU labeling revealed that the nuclei of inseminated eggs (Fig. 2Da), but not uninseminated eggs (Fig. 2Db), incorporate BrdU before the expected time of first

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Fig. 2. Fertilization-induced cessation of sperm attraction (A), expression of surface adhesion (B), and progression of cell cycle (C and D). (A) The number of sperm attracted to a fixed zone near a targeted egg was scored per 10 s following insemination. During the recording period, sperm suspension was repeatedly (every ∼2 min) added to the chamber. The egg ceased sperm attraction and underwent normal cleavage following the recording period. (B) The percentage of eggs showing surface adhesion was continually investigated using a tungsten wire. In respective eggs, the time of fertilization was determined by monitoring the Ca2+ change. Twenty eggs obtained from 3 female jellyfish were observed for each time point. (C) CGD-injected and fertilized eggs were continually observed for the investigation of the timing of cytokinesis or the first cleavage. A minimum of 30 eggs obtained from 4 females were assessed per each time point. A bright-field image (inset) indicates a typical example of 2-cell embryo, which was photographed at 55 min after fertilization. Bar, 50 μm. (D) The eggs inseminated (a) and uninseminated (b) were cultured in the presence of 10 mM BrdU for 30 min and prepared for indirect immunofluorescence for the detection of BrdU incorporation. Bar, 50 μm.

cleavage. The data confirm the DNA synthesis prior to the initiation of cytokinesis in normally fertilized Cladonema eggs. Induction of post-fertilization events in unfertilized eggs by injection of IP3 Injection of IP3 into unfertilized eggs can cause a Ca2+ increase in hydrozoans including Cladonema (Deguchi et al., 2005). We confirmed that Cladonema eggs injected with 10 μM IP3 (estimated final intracellular concentration: 400–800 nM) exhibit a single Ca2+ increase similar to that seen at fertilization (n = 6; Fig. 3A). In the following experiments, we examined whether the IP3 injection can trigger MAP kinase dephosphorylation and/or post-fertilization events. Immunoblot analysis using antibodies against pan-ERK and phosphorylated ERK demonstrated that MAP kinase dephosphorylation already occurs at 10 min post-injection in the IP3injected eggs but not in the control eggs injected with PAH buffer alone (n = 3; Fig. 3B). When the maximum number of attracted sperm for the IP3-injected egg was compared with that for the control egg at 10 min post-injection, the former was invariably lower than the latter in each experiment (6/6; Fig. 3C). Surface adhesion was detected in all of the IP3-injected eggs but not in the control eggs at 10 min post-injection (Fig. 3C). IP3-injected eggs underwent cytokinesis or pseudoclea-

vage at 50–80 min post-injection, whereas there was no morphological change in most of the control eggs (Fig. 3D). Furthermore, IP3-injected eggs incorporated BrdU before the expected time of pseudocleavage (Fig. 3E). These results suggest that the eggs in which a Ca2+ increase is artificially induced by IP3 injection, like fertilized eggs, undergo not only MAP kinase dephosphorylation but also post-fertilization events. Inhibition of post-fertilization events in fertilized eggs by injection of BAPTA We next examined whether an intracellular Ca2+ increase is also necessary for the occurrence of post-fertilization events in Cladonema eggs. Carroll et al. (2000) reported that preinjection of BAPTA/Ca2+ into sea urchin eggs blocks a sperm-induced Ca2+ increase without inhibiting sperm entry into them. When Cladonema eggs were similarly preinjected with BAPTA/Ca2+ and inseminated, a Ca2+ increase was not generated. However, sperm nuclei were not detected in these eggs following the fixation and staining with DAPI (data not shown), suggesting that preinjection of BAPTA/Ca2+ precludes sperm entry itself in Cladonema eggs. Alternatively, we injected BAPTA/Ca2+ into targeted eggs after confirming the initiation of Ca2+ increase and investigated its effects on

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Fig. 3. Induction of Ca2+ increase, MAP kinase dephosphorylation, and post-fertilization events by injection of IP3 into unfertilized eggs. The estimated intracellular concentration of IP3 was 400–800 nM in each experiment. (A) IP3 was injected during the Ca2+ measurement at the time point indicated by an arrow. Following the Ca2+ measurement, the presence of surface adhesion and subsequent pseudocleavage in this egg were confirmed. (B) The eggs injected with IP3 (left) and the control eggs injected with PAH buffer alone (right) were collected at 10 min post-injection and subjected to immunoblots with antibodies against pan-ERK (upper panel) and phosphorylated ERK (lower panel). (C) The sperm-attracting ability and the surface adhesive property were checked at 10 min after injection of IP3 and PAH buffer. In each of the 6 separate experiments, equal amounts of the same sperm suspension were added to IP3- and PAH buffer-injected eggs obtained from the same batch, and the maximum numbers of attracted sperm during a period of 3 min were compared (a pair of filled and open bars in each experiment). Just before the assay for the sperm attraction, each egg was touched with a tungsten wire to evaluate its adhesive property. The marks on the bars indicate the presence (+) or absence (−) of adhesion to the tungsten wire. (D) Time course of cytokinesis or pseudocleavage was examined after injection of IP3 (open circles) and PAH buffer (filled squares). At least 26 eggs obtained from 8 batches were assessed per each time point. A bright-field image (inset) indicates a typical example of pseudocleavage, which was photographed at 60 min after IP3 injection. Bar, 50 μm. (E) The egg injected with IP3 was cultured in the presence of 10 mM BrdU for 40 min and prepared for indirect immunofluorescence for the detection of BrdU incorporation. Bar, 50 μm.

the subsequent events. When BAPTA/Ca2+ was injected into CGD-injected eggs at 8–12 s after fertilization, the Ca2+ elevation was immediately prevented, and the Ca2+ level returned nearly to or slightly below the original value (n = 8; Fig. 4A). Spatial analysis of the Ca2+ change demonstrated that BAPTA/Ca2+ injection is done around the time when the front of the propagating Ca2+ wave is about to reach or just

arrives at the antipode of the wave initiation site (data not shown). The eggs injected with BAPTA/Ca2+ failed to stop sperm attraction subsequently; the maximum number of attracted sperm at 10 min post-fertilization for the BAPTA/ Ca2+-injected egg was invariably higher than that for the control egg injected with PAH buffer alone in each experiment (8/8; Fig. 4B). In addition, surface adhesion was

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Fig. 4. Effects of BAPTA/Ca2+ injection into fertilized eggs on the Ca2+ change and post-fertilization events. BAPTA/Ca2+ was injected into CGD-injected eggs at 8– 12 s (A and B) and 30–40 s (C and D) after the time of fertilization. (A and C) Ca2+ levels in CGD-injected and fertilized eggs were immediately lowered by the injection of BAPTA/Ca2+ (arrows). (B and D) In each experiment, the sperm-attracting ability and the surface adhesive property were checked at 10 min postfertilization for the comparison between the egg injected with BAPTA/Ca2+ and the control egg injected with PAH buffer alone at the same time after fertilization. The experimental methods and the mode of presentation are the same as those in Fig. 3C. The data indicated by the filled (left) bars in Exp. 5 of panel B and Exp. 2 of panel D are obtained from the eggs used for panels A and C, respectively.

detected not in the BAPTA/Ca2+-injected eggs but in the control eggs (Fig. 4B). In contrast to the above situation, slightly later injection of BAPTA/Ca2+ generated completely different results. When the same BAPTA/Ca2+ injection was performed at 30–40 s after fertilization, the Ca2+ level was similarly lowered (n = 6; Fig. 4C). Under these conditions, however, the sperm-attracting ability of BAPTA/Ca2+-injected egg was reduced, similar to the control egg injected with PAH buffer alone (Fig. 4D). In addition, the BAPTA/Ca2+ and the control injections at 30–40 s after fertilization both allowed the recipient eggs to express the surface adhesion (Fig. 4D). These results suggest that a Ca2+ increase lasting for 15–30 s is necessary for cessation of sperm attraction and expression of surface adhesion. However, it was unclear whether the Ca2+ increase is also a necessary signal for progression of cell cycle, since cytokinesis or cleavage was constantly inhibited in BAPTA/Ca2+-injected eggs, independently of the time of injection following fertilization. It is known in sea urchin eggs that injection of BAPTA/Ca2+ gives some toxic effect on the overall health of the recipient eggs (Carroll et al., 2000). It might also be possible that chelation of Ca2+ inhibits much later Ca2+-dependent events, such as nuclear envelope breakdown (Wilding et al., 1996).

Induction of post-fertilization events in unfertilized eggs by treatment with U0126 The fact of rapid MAP kinase dephosphorylation after fertilization or IP3 injection led us to assume that this is a necessary and/or sufficient step linking a Ca2+ increase to the subsequent events in Cladonema eggs. In this series of experiments, we examined effects of a potent and selective MEK inhibitor, U0126 (Favata et al., 1998; English et al., 1999), which is known to cause MAP kinase dephosphorylation in oocytes or eggs of various marine invertebrates (Levasseur and McDougall, 2000; Kumano et al., 2001; Harada et al., 2003; Portillo-López et al., 2003). The addition of 20 μM U0126 to unfertilized eggs did not induce a Ca2+ increase at least during a recording period of ∼30 min (n = 4; Fig. 5A). Immunoblot analysis demonstrated that the signal of phosphorylated form of MAP kinase decreases gradually and disappears eventually at 25–30 min post-treatment, although the total amount of MAP kinase is relatively constant (n = 3; Fig. 5B). Consistent with the time course of MAP kinase dephosphorylation, U0126-treated eggs retained the sperm-attracting ability and failed to express surface adhesion during the first 20-min period (data not shown). However, the sperm-attracting ability of U0126-treated

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Fig. 5. Induction of MAP kinase dephosphorylation and post-fertilization events, but not Ca2+ increase, by application of U0126 to unfertilized eggs. The final concentration of U0126 was 20 μM in each experiment. (A) U0126 was added during the Ca2+ measurement at the time point indicated by an arrow. Following the Ca2+ measurement, the presence of surface adhesion and subsequent pseudocleavage in this egg were confirmed. (B) Samples of U0126-treated eggs were collected every 5 min following stimulation and subjected to immunoblots with antibodies against pan-ERK (upper panel) and phosphorylated ERK (lower panel). (C) The spermattracting ability and the surface adhesive property were checked at 40 min after application of U0126 and DMSO alone (control). The experimental methods and the mode of presentation are the same as those in Fig. 3C. (D) Time course of cytokinesis or pseudocleavage was examined after application of U0126 (open circles) and DMSO (filled squares). At least 14 eggs obtained from 3 batches were assessed per each time point. A bright-field image (inset) indicates a typical example of pseudocleavage, which was photographed at 95 min after U0126 application. Bar, 50 μm. (E) The eggs treated with U0126 (a) and DMSO (b) were cultured in the presence of 10 mM BrdU for 60 min and prepared for indirect immunofluorescence for the detection of BrdU incorporation. Bar, 50 μm.

egg became lower than that of DMSO-treated control egg by 40 min post-treatment (8/8; Fig. 5C). At this time, U0126treated eggs, but not control eggs, showed surface adhesive property (Fig. 5C). Furthermore, U0126-treated eggs, but not control eggs, eventually underwent cytokinesis or pseudocleavage (Fig. 5D), similar to the case in IP3-injected eggs (Fig. 3D). It should be noted that the timing of cytokinesis induced by U0126 treatment is also later than that induced by IP3 injection (compare Fig. 5D with Fig. 3D), probably due to the gradual MAP kinase dephosphorylation. In BrdU labeling experiments, fluorescent signals for BrdU incorporation were detected in the nuclei of eggs treated with 20 μM U0126 (Fig. 5Ea) but not with DMSO (Fig. 5Eb), indicating that DNA synthesis is actually caused in U0126-treated eggs. It is, therefore, likely that cytokinesis or pseudocleavage in U0126-treated eggs is the phenomenon accompanied by progression of cell cycle, as is the case in those eggs that are activated by a Ca2+ increase. Neither surface adhesion nor cytokinesis took place in the eggs treated with U0124, an inactive analogue of U0126, at 20 or 100 μM (data not shown). Furthermore, a non-specific toxic

effect of U0126 may be ruled out, since the eggs inseminated at 10 min after application of 20 μM U0126 and cultured in the continuous presence of this drug exhibited normal mitosis; 11 out of the 12 eggs developed to normal 2-cell embryos at 40– 70 min post-insemination. These results collectively suggest that MAP kinase dephosphorylation by U0126 treatment is a sufficient trigger for the post-fertilization events, although somewhat longer time is required for these processes. Inhibition of post-fertilization events in fertilized eggs by injection of GST-Mos In the final series of experiments, we tested effects of the GST fusion protein of Mos, which is expected to activate MAP kinase phosphorylation pathway persistently (Tachibana et al., 2000) and therefore prevent the MAP kinase dephosphorylation after fertilization. Preinjection of GST-starfish Mos (estimated final intracellular concentration: 20–40 μg/ml) into Cladonema eggs did not inhibit a Ca2+ increase after insemination; a normal range of peak Ca2+ levels (F/F0 > 1.4) was detected in most of

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the eggs tested (7/11; Fig. 6A), although peak values were relatively low (F/F0 < 1.4) in the remaining 4 eggs. As expected, MAP kinase dephosphorylation following insemination was inhibited in the eggs preinjected with GST-Mos but not in the control eggs preinjected with GST alone (Fig. 6B). In GST-Mos-injected eggs, post-fertilization events were severely affected even in the above 7 eggs showing a normal Ca2+ increase after insemination. First, the maximum number of attracted sperm examined at 10 min post-fertilization for the GST-Mos-injected egg was invariably higher than that for the control egg preinjected with GST alone in each experiment (7/7; Fig. 6C). Second, surface adhesion was detected not in the GSTMos-injected eggs but in the control eggs (Fig. 6C). Finally, none of the GST-Mos-injected eggs (0/7) underwent cytokinesis for at least 120 min following fertilization, whereas all of the control eggs (7/7) exhibited the normal first cleavage at 50– 80 min post-fertilization. Consistent with the absence of cytokinesis, BrdU incorporation following fertilization, which was detected in the control eggs (Fig. 6Db), was prevented in GST-Mos-injected eggs (Fig. 6Da). These data strongly suggest

that MAP kinase dephosphorylation is necessary for the occurrence of post-fertilization events in fertilized eggs. Discussion From the results presented here, we made a model for the regulation of post-fertilization events by Ca2+ increase via MAP kinase dephosphorylation in Cladonema eggs (Fig. 7). Fertilization and IP3 injection both cause an intracellular Ca2+ increase, trigger immediate MAP kinase dephosphorylation, and induce post-fertilization events including cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle leading to DNA synthesis and cytokinesis. Injection of BAPTA/Ca2+ into fertilized eggs, which immediately lowers the elevated Ca2+ level to the resting value, suppresses the post-fertilization events when performed at 8– 12 s post-fertilization. Induction of MAP kinase dephosphorylation by U0126 triggers the post-fertilization events without producing a Ca2+ increase. Conversely, inhibition of MAP kinase dephosphorylation by GST-Mos prevents all of the post-

Fig. 6. Effects of preinjection of GST-Mos on the sperm-induced Ca2+ and MAP kinase changes and post-fertilization events. GST-starfish Mos and the control protein (GST alone) were injected into unfertilized eggs at 3–5 min before insemination. The estimated intracellular concentrations of these proteins were 20–40 μg/ml. (A) The GST-Mos-injected egg was inseminated at time zero and subjected to the Ca2+ measurement subsequently. No cleavage was observed in this egg following the Ca2 + measurement. (B) The eggs preinjected with GST-Mos (left) and the control eggs preinjected with GST alone (right) were collected at 10 min post-insemination and subjected to immunoblots with antibodies against pan-ERK (upper panel) and phosphorylated ERK (lower panel). (C) In each experiment, the sperm-attracting ability and the surface adhesive property were checked at 10 min after fertilization for the comparison between the egg injected with GST-Mos and the control egg injected with GST alone. The experimental methods and the mode of presentation are the same as those in Fig. 3C. The data indicated by the filled (left) bar in Exp. 4 are obtained from the egg used for panel A. (D) The eggs preinjected with GST-Mos (a) and GST alone (b) were cultured in the presence of 10 mM BrdU for 40 min following insemination. They were then prepared for indirect immunofluorescence for the detection of BrdU incorporation. Bar, 50 μm.

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Fig. 7. A model for the regulation of post-fertilization events by Ca2+ increase and MAP kinase dephosphorylation in Cladonema eggs. A fertilization-induced intracellular Ca2+ increase, which can be mimicked by IP3 and inhibited by BAPTA/Ca2+, dephosphorylates MAP kinase. MAP kinase dephosphorylation, which can be artificially induced by U0126 and suppressed by GST-Mos, then triggers post-fertilization events including cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle leading to DNA synthesis and cytokinesis.

fertilization events even in the presence of Ca2+ increase. These data strongly suggest that a Ca2+ increase triggers MAP kinase dephosphorylation, which in turn causes the post-fertilization events in Cladonema eggs. Changes in intracellular Ca2+ level and MAP kinase activity at fertilization It is known that fertilized oocytes or eggs exhibit intracellular Ca2+ increases, regardless of their meiotic stages arrested prior to fertilization (Stricker, 1999). On the other hand, the states of MAP kinase in unfertilized oocytes or eggs, as well as its changes after fertilization, seem to be different, depending upon the stages of fertilization. In such animals as echiuran worms (Gould and Stephano, 1999; Tan et al., 2005) and some bivalves (Shibuya et al., 1992; Stephano and Gould, 2000), where fertilization takes place at the first prophase of meiosis, MAP kinase is maintained in the dephosphorylated or inactive form in unfertilized oocytes but phosphorylated or activated after fertilization or artificial stimulation. In contrast, phosphorylated or active MAP kinase is already present before fertilization in the first or second metaphase-arrested oocytes of ascidians (Russo et al., 1996; McDougall and Levasseur, 1998), nemertean worms (Stricker and Smythe, 2003), frogs (Watanabe et al., 1991), and mammals (Verlhac et al., 1994; Moos et al., 1995; Liu and Yang, 1999); in these cases, fertilization or artificial stimulation results in MAP kinase dephosphorylation or inactivation. In eggs arrested at the pronuclear stage, it has been demonstrated that phosphorylated or active MAP kinase is also present before fertilization, and that its dephosphorylation or inactivation occurs after fertilization or artificial stimulation in most studies using starfish (Abrieu et al., 1997; Tachibana et al., 1997; Sadler and Ruderman, 1998; Fisher et al., 1998) and sea urchins (Chiri et al., 1998; Carroll et al., 2000; Kumano et al., 2001; Zhang et al., 2005). Our present data suggest a similar situation in the more

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primitive animal, hydrozoan jellyfish. However, absolutely different behavior of MAP kinase during fertilization is detected in some studies using sea urchin eggs (Philipova and Whitaker, 1998; Philipova et al., 2005). Immunohistological analyses reveal that phosphorylated or active MAP kinase is distributed in both the cytoplasm and the nucleus in sea urchin eggs before fertilization (though much concentrated in the nucleus), and that the nuclear and cytoplasmic signals both disappear after fertilization (Kumano et al., 2001; Zhang et al., 2005). The influence of such subcellular localization of MAP kinase, as well as the possibility of existence of multiple MAP kinases, may explain the contradictory reports for sea urchin eggs (see Zhang et al., 2005). The state of MAP kinase is determined by a balance between activities of the upstream Mos/MEK/MAP kinase cascade that continuously provides phosphorylated MAP kinase and those of phosphatase(s) responsible for the direct dephosphorylation of MAP kinase (see Kumano et al., 2001). Degradation of Mos protein and deadenylation of mos mRNA seem to be the main mechanisms responsible for MAP kinase dephosphorylation in fertilized frog oocytes (reviewed by Tunquist and Maller, 2003), where associated phosphatase activities responsible for MAP kinase inactivation remain constant (Sohaskey and Ferrell, 1999). In contrast, it is suggested in sea urchin eggs that inactivation/inhibition of the upstream cascade and activation of the phosphatase(s) are both responsible for the MAP kinase dephosphorylation after fertilization (Kumano et al., 2001). In Cladonema eggs, MAP kinase dephosphorylation was able to be induced by application of U0126 at a concentration of 20 μM or higher but not adequately by 10 μM (data not shown). The effective concentration of U0126 (20 μM) is within the range of the dose used for other invertebrates (10 μM for ascidian oocytes, Levasseur and McDougall, 2000; 50 μM for oyster and mussel oocytes, Portillo-López et al., 2003; 100 μM for starfish oocytes, Harada et al., 2003), although much lower concentrations (0.5–1 μM) are sufficient to cause MAP kinase dephosphorylation in sea urchin eggs (Kumano et al., 2001; Zhang et al., 2005). The process of MAP kinase dephosphorylation in U0126-treated eggs required longer time than that in fertilized and IP3-injected eggs in Cladonema. One possible explanation for the delay in MAP kinase dephosphorylation following U0126 treatment is its gradual or insufficient permeability across the plasma membrane. However, this possibility may not solely explain such a situation, since the timing of post-fertilization events was not altered when U0126 was used at 100 μM, instead of 20 μM (data not shown). Alternatively, activation of phosphatase might be the dominant pathway responsible for the MAP kinase dephosphorylation in fertilized Cladonema eggs. If this is the case, phosphatase activities would be rapidly enhanced in a Ca2+-dependent manner. It is shown in mammalian fibroblasts that Ca2+-dependent activation of MAP kinase phosphatase-1 (MKP-1) plays a role in the regulation of ERK2 (Plows et al., 2002). Ca2+-dependent stimulation of phosphatase activities responsible for MAP kinase dephosphorylation is also detected in lysates of sea urchin eggs (Kumano et al., 2001).

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We also found that MAP kinase dephosphorylation in fertilized Cladonema eggs is suppressed by preinjection of GST-Mos at a final intracellular concentration of 20–40 μg/ml. Similar concentrations of GST-Mos in starfish oocytes (∼20 μg/ ml, Tachibana et al., 2000) and MBP (maltose-binding protein)Mos in frog oocytes (∼10 μg/ml, Murakami and Vande Woude, 1998) maintain elevated levels of MAP kinase activity. These fusion proteins are thought to be the non-degradable and constitutively active forms of Mos; it is shown in frog oocytes that MBP-Mos is not degraded following activation by Ca2+ ionophore, while endogenous Mos is degraded normally in the presence of MBP-Mos (Murakami and Vande Woude, 1998). We consider it likely that GST-Mos similarly persists in fertilized Cladonema eggs, overcoming the Ca2+-regulated downstream pathways leading to MAP kinase dephosphorylation. The mechanisms and associated molecules linking a Ca2+ increase and MAP kinase dephosphorylation in fertilized hydrozoan eggs should be clarified in future studies. Progression of cell cycle triggered by Ca2+ increase and MAP kinase dephosphorylation The phenomenon of pseudocleavage has been observed in the eggs activated by Ca2+ ionophore in the hydrozoan Phialidium (Freeman and Ridgway, 1993). We induced this morphological change in Cladonema eggs by injection of IP3, a physiological activator of Ca2+ release (Deguchi et al., 2005). The pseudocleavage seems to occur as the result of cell cycle progression, since it is preceded by DNA synthesis, which can be detected by incorporation of [3H]-thymidine (Freeman and Ridgway, 1993) and BrdU (this study) into nuclei. These data strongly suggest that a Ca2+ increase works as a trigger for G1/S transition in hydrozoan eggs, as is the case in sea urchin eggs (Carroll et al., 2000; Philipova et al., 2005). Our present study also demonstrated that DNA synthesis and subsequent cytokinesis can be induced by 20 or 100 μM U0126. In sea urchin eggs, the dose effect of U0126 and PD 98059, a less effective MEK inhibitor, is becoming the target of debate. The use of 20 μM PD 98059 causes DNA synthesis without insemination (Carroll et al., 2000), while 100 μM U0126 inversely blocks DNA synthesis following fertilization (Philipova et al., 2005). Zhang et al. (2005) pointed out non-specific toxic effects of high concentrations of MEK inhibitors (>50 μM) on sea urchin eggs. In Cladonema, insemination to U0126-treated eggs resulted in normal cleavage, instead of pseudocleavage, in the continuous presence of the drug. In addition, the control chemical U0124 was completely ineffective in triggering pseudocleavage even at 100 μM. Thus, we feel that the effect of U0126 is rather specific, and the concentrations we used have no considerable toxicity on Cladonema eggs. However, we cannot eliminate the possibility that the molecules other than MEK are also influenced in U0126-treated Cladonema eggs. We also found that suppression of MAP kinase dephosphorylation by preinjection of GST-Mos completely prevents DNA synthesis and cytokinesis in fertilized Cladonema eggs. The existence of GST-Mos might have some inhibitory effect

on Ca2+ change itself, since peak Ca2+ levels at fertilization were lowered in some GST-Mos-injected eggs, while such a situation was not detected in the control eggs injected with GST alone (data not shown). Nevertheless, the post-fertilization events were indeed blocked by GST-Mos even in the eggs showing a normal Ca2+ increase. Thus, it is unlikely that the inhibitory effect of GST-Mos on cell cycle progression is derived from the suppression of Ca 2+ levels. The above results collectively support the view that MAP kinase dephosphorylation is a necessary and sufficient signal for G1/S transition in Cladonema eggs, as suggested in starfish (Tachibana et al., 1997) and sea urchin eggs (Carroll et al., 2000). In mammalian somatic cells such as fibroblasts, ERK is known to regulate G1/S transition both positively and negatively, depending upon the extent of ERK activation (reviewed by Roovers and Assoian, 2000; Zhang and Liu, 2002); a robust early phase activation of ERK followed by a less robust sustained phase leads to expression of cyclin D1, accumulation of active cyclin D1-cdk4 and cyclin E-cdk2 complexes, and G1 phase progression toward S phase, whereas a robust and persistent activation of ERK results in long-term induction of p21cip1, which acts as an inhibitor of cyclin-cdk activity and arrests the cell cycle at G1 phase. In either case, initial targets of ERK involve transcription factors regulating the expression of cell cycle-associated proteins (Roovers and Assoian, 2000; Zhang and Liu, 2002). In contrast, inhibition of protein synthesis does not prevent (Hinchcliffe et al., 1998), or rather, can trigger (Tachibana et al., 1997) G1/S transition in fertilized echinoderm eggs. Cyclin E/cdk2 levels and activities, which increase before G1/S transition and play an essential role in this process in mammalian somatic cells (Kahl and Means, 2003), are already high in unfertilized sea urchin eggs arrested at G1 phase (Sumerel et al., 2001). In addition, G1/S transition cannot be suppressed when cdk activities are inhibited (Moreau et al., 1998). It seems, therefore, likely that the first entry into S phase in echinoderm eggs is regulated mainly by posttranslational modification, which is quite different from the situation in somatic cells. In our preliminary experiment, application of the protein synthesis inhibitor emetine (at 100 μM) to unfertilized Cladonema eggs is effective in inducing cytokinesis or pseudocleavage (Kondoh and Deguchi, unpublished data), implying that similar cell cycle regulatory mechanisms are present in hydrozoan and echinoderm eggs. Cessation of sperm attraction and expression of surface adhesion triggered by Ca2+ increase and MAP kinase dephosphorylation Sperm-attracting substances are thought to be released from the oocytes or eggs themselves in most hydrozoans (Miller, 1978, 1979; Freeman, 1987; Freeman and Miller, 1982) as well as in ascidians (Yoshida et al., 1993). In Cladonema eggs, there is no egg membrane or chorion outside the plasma membrane, and the only extracellular structure covering them is the thin jelly layer (Yamashita, 1987). Since removal of the jelly layer in acid seawater or Ca2+, Mg2+-free seawater did not affect the egg's ability for sperm attraction (data not

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shown), Cladonema eggs may also release sperm-attracting substances by themselves. Miller (1978) reported that sperm attraction stops at 11 min following insemination in eggs of the hydrozoan Orthopyxis. Similar rapid cessation of sperm attraction in fertilized eggs is also observed in the different hydrozoan Phialidium (Freeman and Miller, 1982). However, quantitative analysis of this phenomenon has not yet been done in these species. In the present study using Cladonema, we clarified, for the first time, the temporal change in the number of sperm attracted by a single egg. The egg's ability for sperm attraction began to decrease within a few minutes of fertilization and eventually vanished by an additional 5 min. The time course of the attenuation of sperm attraction in fertilized eggs is consistent with the view that MAP kinase dephosphorylation acts as a trigger for this event (see below). The gradual decrease in sperm attraction would be due to gradual reduction of sperm attractant released from the eggs. The jelly layer surrounding the eggs might also play a supplementary role in the gradual loss of sperm attraction, since the structure can act like a chromatographic matrix, delivering the attractant to the external medium more slowly than normal diffusion (Miller, 1985). Our present results strongly suggest that a Ca2+ increase is a necessary as well as sufficient signal for cessation of sperm attraction and that MAP kinase dephosphorylation also plays an essential role in the step linking a Ca2+ increase to this event. It seems likely that once MAP kinase dephosphorylation is triggered by a brief Ca2+ increase lasting for 15–30 s, the molecule(s) involved in the following pathways can function without elevated Ca2+ levels. In ascidian oocytes, the ability for sperm attraction has been lost by the time of cortical contraction, which takes place at ∼2 min after the onset of initial Ca2+ wave at fertilization (Yoshida et al., 1993). This observation raises the possibility that the cessation of sperm attraction is also regulated by an initial and brief Ca2+ increase in ascidian oocytes. In the case of ascidian oocytes, however, MAP kinase activity slightly increases rather than decreases during the first 10-min period after fertilization (Russo et al., 1996; McDougall and Levasseur, 1998; Marino et al., 2000). The chemical natures of sperm-attracting substances may also be different in hydrozoans (considered to be peptides, see Introduction) and ascidians (identified as a sulfated steroid, Yoshida et al., 2002). In various animals, fertilized oocytes or eggs modify the structural and chemical features of extracellular coat around them. One of the best known cortical changes is exocytosis of cortical granules (CGs), the membrane-bound and secretory vesicles (Wessel et al., 2001). However, neither the existence of apparent CGs underneath the plasma membrane nor the exocytosis of them can be detected in Cladonema eggs by electron microscopic analysis (Yamashita, 1987). Nevertheless, at least some cortical change would be expected to occur, considering the expression of adhesive property after fertilization (Yamashita, 1987; this study). In fertilized mouse oocytes, secretion of the vesicles that are distinct from CGs seems to be responsible for an increase in the amount of calreticulin, a protein that can serve as a mediator of adhesion (Coppolino and

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Dedhar, 1999; Coppolino et al., 1997), on the extracellular surface of the plasma membrane (Tutuncu et al., 2004). In fertilized Cladonema eggs, surface adhesive property began to be detected around the time of the termination of sperm attraction (at ∼6 min post-fertilization). The expression of surface adhesion, as well as the cessation of sperm attraction, was triggered by MAP kinase dephosphorylation downstream of Ca2+ increase. In echinoderm eggs, formation of fertilization membrane resulting from CG exocytosis is a Ca2+-regulated event (reviewed by Jaffe et al., 2001), which can be induced by IP3 (Swann and Whitaker, 1986; Iwasaki et al., 2002) and can be inhibited by BAPTA/Ca2+ (Carroll et al., 2000). However, formation of fertilization membrane is unlikely to depend upon MAP kinase activity, since this event cannot be induced by inactivation of MAP kinase (Tachibana et al., 2000; Carroll et al., 2000) and cannot be inhibited in fertilized eggs under the condition that MAP kinase is constitutively activated (Tachibana et al., 1997). Thus, cortical modifications may be regulated by entirely different mechanisms in hydrozoan and echinoderm eggs. In summary, our present study offers the evidence that an intracellular Ca2+ increase causes post-fertilization events, cessation of sperm attraction, expression of surface adhesion, and cell cycle progression, via MAP kinase dephosphorylation in Cladonema eggs. It will be interesting in future studies to clarify how MAP kinase regulates various cellular events in eggs of the evolutionarily old metazoans, hydrozoans. Acknowledgment This work was supported by a Grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (16770055 to R.D.). References Abrieu, A., Fisher, D., Simon, M., Dorée, M., Picard, A., 1997. MAPK inactivation is required for the G2 to M-phase transition of the first mitotic cell cycle. EMBO J. 16, 6407–6413. Abrieu, A., Dorée, M., Fisher, D., 2001. The interplay between cyclin-B-Cdc2 kinase (MPF) and MAP kinase during maturation of oocytes. J. Cell Sci. 114, 257–267. Agell, N., Bachs, O., Rocamora, N., Villalonga, P., 2002. Modulation of the Ras/ Raf/MEK/ERK pathway by Ca2+, and calmodulin. Cell. Signalling 14, 649–654. Carré, D., Sardet, C., 1981. Sperm chemotaxis in siphonophores. Biol. Cell 40, 119–128. Carroll, D.J., Albay, D.T., Hoang, K.M., O'Neill, F.J., Kumano, M., Foltz, K.R., 2000. The relationship between calcium, MAP kinase, and DNA synthesis in the sea urchin egg at fertilization. Dev. Biol. 217, 179–191. Chiri, S., De Nadai, C., Ciapa, B., 1998. Evidence for MAP kinase activation during mitotic division. J. Cell Sci. 111, 2519–2527. Collins, A.G., 2002. Phylogeny of Medusozoa and the evolution of cnidarian life cycles. J. Evol. Biol. 15, 418–432. Coppolino, M.G., Dedhar, S., 1999. Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events. Biochem. J. 340, 41–50. Coppolino, M.G., Woodside, M.J., Demaurex, N., Grinstein, S., St-Arnaud, R., Dedhar, S., 1997. Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion. Nature 386, 843–847.

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